Weaver Granule Neurons Are Rescued by Calcium Channel Antagonists and Antibodies Against a Neurite Outgrowth Domain of the B2 Chain of Laminin

The weaver mutation impairs migration of the cerebellar granular neurons and induces neuronal death during the first two weeks of postnatal life. To elucidate the molecular mechanisms for the impaired neuronal migration, we investigated the rescue mechanisms of the weaver (wv/wv) granule neurons in vitro. We found that Fab2 fragments of antibodies against a neurite outgrowth domain of the B2 chain of laminin enhanced neurite outgrowth and neuronal migration of the weaver granule neurons on a laminin substratum and in the established cable culture system. The rescue of the weaver granule neurons by antibodies against the B2 chain of laminin may result from the neutralizing effect of these antibodies against the elevated B2 chain levels of the weaver brain. The L-type calcium channel blocker, verapamil (1-5 ixM), also rescued the weaver granule neurons. High concentrations of MK-801 (1020 ixM), a glutamate receptor antagonist and voltagegated calcium channel blocker, rescued the weaver granule neurons similar to verapamil, but low concentrations of MK-801 (1 ~M) had no rescue effect. Simultaneous patch-clamp studies indicated that the weaver granule neurons did not express functional N-methyl-Daspartate receptors further indicating that the rescue of the weaver granule neurons by MK-801 resulted from its known inhibition of voltage-gated calcium channels. The present results indicate that antibodies against the B2 chain of laminin, verapamil, and high concentrations of MK-801 protect the weaver granule neurons from the otherwise destructive action of the weaver gene. Thus, both the laminin system and calcium channel function contribute to the migration deficiency of the weaver granule neurons. T HE weaver mutant mouse provides a model system for studying mechanisms of neuronal migration and death (Sidman et al., 1965), because the granule neurons of the homozygous weaver (wv/wv) mouse cerebellum fail to migrate and die during the first two weeks of postnatal life (Rakic and Sidman, 1973 a,b,c; Sotelo and Changeux, 1974; Sotelo, 1975). The weaver gene defect has been genetically mapped to chromosomel6 in the mouse (Reeves et al., 1989), but the affected gene(s) have not been identified. It has recently been suggested that a point mutation in a GIRK2 K ÷ channel gene (Lesage et al., 1994) could be responsible for the weaver mutation (Patil et al., 1995; Slesinger et al., 1996). However, recent electrophysiological experiments contradict this view, because GIRK2 channel activity was detected neither in normal (+ /+) nor weaver (wv/wv) granule neurons during the developmental stage when the neurons die (Mjaatvedt et al., Address all correspondence to Dr. P~iivi Liesi, LMCN, NIAAA, NIH, 12501 Washington Avenue, Rockville, MD 20852. Tel.: (301) 443-2456. Fax: (301) 443-5894. E-mail: [email protected] 1995). Genetic studies indicate that the weaver defect is intrinsic to the granule neurons (Goldowitz and Mullen, 1982; Goldowitz, 1989), and recent in vitro and transplantation studies have further shown that the weaver granule neurons can be rescued by normal granule neurons (Gao and Hatten, 1993) or their membrane extracts (Gao et al., 1992). The role of calcium channel function and glutamate receptors in the migratory failure and death of the weaver granule neurons has not been investigated even though recent evidence indicates that normal migratory cerebellar granule neurons express functional N-methyl-o-aspartate (NMDA) 1 receptors (Rossi and Slater, 1993; Farrant et al., 1994), and that both NMDA receptor blockers and voltage-gated calcium channel antagonists inhibit neuronal migration of the normal cerebellar granule neurons (Komuro and Rakic, 1992, 1993). Elevated levels of intracellular calcium are involved in neurodegenerative mechanisms of the brain tissue (Choi, 1988) and it is therefore of spe1. Abbreviat ions used in this paper: GAD, glutamic acid decarboxylase; GFAP, glial fibrillary acidic protein; NMDA, N-methyl-D-aspartate. © The Rockefeller University Press, 0021-9525/96/07/477/10 $2.00 The Journal of Cell Biology, Volume 134, Number 2, July 1996 477486 477 on Jne 7, 2017 D ow nladed fom Published July 15, 1996